The goal of this proposal is to determine how membrane trafficking of critical Notch pathway components during asymmetric cell division controls a Notch-mediated cell fate switch in cells of the nervous system. Notch function is required in a variety of normal stem and progenitor cells, including neural stem cells, blood cell precursors, and gut progenitors in both Drosophila and vertebrates. Aberrant Notch regulation in stem cells leads to severe defects in developmental events and contributes to human diseases, such as CADASIL syndrome, and cancers, such T-cell acute lymphoblastic leukemia. To understand in detail the cell biological mechanisms that control Notch function, the proposal focuses on the sensory organ progenitor (SOP) cells of the Drosophila adult peripheral nervous system, where cell fate decisions depend on activation or inhibition of Notch activity. The work is based on the findings over the last five years that establish the functional role of a key regulato of Notch signaling in sensory organ cells, Sanpodo. In the previous funding cycle, studies demonstrated that sanpodo promotes Notch activity to confer correct cell fates after asymmetric cell division in the adult peripheral nervous system. Through biochemical studies, in vivo imaging, and rescue experiments, functional domains of Sanpodo have been identified that mediate two critical functions. First, Sanpodo associates with the g-secretase complex to promote Notch activation in the cell with high Notch activity. Second, Sanpodo controls Notch recycling at the plasma membrane in the cell with low Notch activity, acting as a repressor of Notch signaling in this context. This proposal aims to determine how these antagonistic functions are regulated at the level of vesicle trafficking and to define the role of conserved motifs that determine Sanpodo's interaction with Notch regulators such as Numb. The role of conserved vesicle trafficking regulators on controlling Notch levels in asymmetrically dividing neural progenitors will be assessed, using the unique ability to combine molecular modeling, biochemical analysis, and live cell imaging of progenitor cell behavior in the intact animal, to tet the hypothesis that a constitutive level of endocytosis establishes steady state levels of Notch in epithelial cells, whereas Sanpodo acts synergistically with Numb to deplete Notch from the plasma membrane in asymmetrically diving neural progenitors. This proposal will therefore provide a more complete understanding of the evolutionarily conserved mechanisms governing Notch trafficking in this context.